Name: Felicia Reeves ID #: 1104148 Date: September 24,2012 Title: Aim: The development of and use of the Cyanmethaemoglobin

in Standard curve To determine the Haemoglobin concentration of an EDTA sample using Cyanmethaemoglobin standard curve To perform a 1/250 dilution on a portion of EDTA blood To determine the PCV of the EDTA Sample

Principle: Whole blood is diluted in cyanmethemoglobin reagent. This reagent hemolyzes erythrocytes, which releases hemoglobin into the solution. The ferrous ions (Fe2+) of the hemoglobin molecules are oxidized by potassium ferricyanide to ferric ions (Fe 3+). This oxidation results in the formation of methemoglobin. Methemoglobin combines with the cyanide ions(CN-) to form cyanmethemoglobin, a stable compound. All hemoglobin derivatives except sulfhemoglobin are converted to cyanmethemoglobin. When measured spectrophotometrically at 540nm,the absorbance of cyanmethemoglobin follows Lambert-Beers law and is directly proportional to the concentration of hemoglobin in the blood. A reference (standard) curve is prepared using cyanmethemoglobin standard solutions of known hemoglobin concentrations. An unknown hemoglobin concentration may be calculated from the measured absorbance,read from

the calibration curve. (Prenhall n.d) Packed cell volume (PCV) is a measure of the proportion of blood volume that is occupied by red blood cells. The normal sample range for female is (35-39)L/L, and male (40-54) L/L. Rule of Three PCV is 3 times the hb for normal individuals. Mean Cell Hemoglobin Concentration (MCHC) normal sample range (31-36) g/dl. Measures the average Hb concentration in the RBCs Wright's stain is a Romanowsky type metachromatic stain made by mixing old or specially treated methylene blue dye with eosin in a methanol diluent. Basic components of the cell, such as hemoglobin or certain inclusions or granules, will unite with the acidic portion of the stain, eosin, and are said to be eosinophilic. These components are stained varying shades of pink or red. Acidic cell components, such as nucleic acids, reactive cytoplasm, etc. take up the basic dye components, methylene azure, and stain blue or purple. pH must be carefully controlled through the use of a buffer of 6.4-6.7. If the pH is too acidic the stain will take on a pinkish tint, and nuclear structures will be poorly stained. A basic pH will cause all intracellular structures, nuclei, etc. to be blue-black in color, with poorly defined structure. (The Science advisory board 2004)

Methodology: 1. Developing the Cyanmethaemoglobin Standard Curve Dilutions of Cyanmethaemoglobin (amber-coloured solution) were made with Drabkins reagent (straw-yellow coloured solution) in a test tube. The test tubes were covered with parafilm and mixed well by inversion The spectrophotometer was ready for use at 540nm

The dilutions made were transferred to clean glass cuvettes The absorbance values of these solutions were read on the spectrophotometer at 540nm

The readings obtained were recorded A graph was plotted of Haemoglobin Concentration(g/dl) on the abscissa against absorbance (optical density) on the ordinate

The line of best fit was drawn

2. Perform a 1/250 dilution on the portion of EDTA sample provided 5ml of Drabkins was pipette 0.02 ml of EDTA blood was pipette The solution was mixed well by inversion and incubated for 3 minutes at room temperature The absorbance was read on the spectrophotometer (540 nm).

3. Using the Cyanmethaemoglobin standard curve, the haemoglobin concentration of the EDTA sample was determined 4. The PCV of the EDTA sample was determined 5. The MCHC of the EDTA sample was calculated 6. All results obtained were recorded

7. The refence ranges provided were used to comment on the results obtained for the EDTA sample.

Results: Tube 1 2 3 4 5 Sample A Absorbance 0.594 0.475 0.320 0.167 0.038 0.414 Concentration (g/dl) 20 15 10 5 0 13

Hb concentration from graph = 13 g/dl Cu= Au/As * Cs 0.414/0.594 * 20 = 13.9 g/dl

MCHC= Hb/PCV 13.9/O.40 = 34.8 g/dl

Conclusion: The hemoglobin concentration of the sample from the graph was 13 g/dl, and when calculated was 13.9 g/dl. These values were not the same, however they were similar. The Hb concentration for the sample fit within the reference range which is (12-16) g/dl for females and (14-18) g/dl for males.

The PCV determined was 0.40 L/L and the normal ranges from (0.35-0.39) L/L ,the PCV for the sample does not fall in this range meaning this is not normal. PCV is 3 times the Hb for normal individuals. The MCHC when calculated was 34.8 this falls within the normal reference range which is (3136) g/dl